Project Summary/Abstract Effects of Brca1 Heterozygosity on Mammary Gland Biology Germline heterozygous BRCA1 mutations hugely predispose women to breast and ovarian cancer; however, the basis for the tissue specificity of this effect remains obscure. Burgeoning evidence suggests that this gene?s mutation may cause phenotypic changes in mammary tissue prior to tumor formation. Uncovering the precise nature and mechanisms of the changes in the precancerous breast will prove vital to developing improved preventative treatments for this familial cancer syndrome. Unfortunately, clinical progress in this field is stymied by disagreements in the scientific literature concerning which mammary populations are perturbed; indeed, this issue is a subcase of the broader ambiguity surrounding the cellular composition of the normal mammary gland. In this proposal, we describe plans to use mass cytometry to systematically test our hypothesis that Brca1 heterozygosity induces the aberrant accumulation of one or more mammary cell types and that these populations display altered differentiation and DNA repair phenotypes which ultimately make them more tumorigenic than other mammary cells. In Aim 1, I will analyze mammary cell populations using single-cell proteomics via mass cytometry, which will enable a high-resolution understanding of the cell types present throughout normal mammary gland development and will allow a more precise understanding of the nature of alterations in the development and premalignant phenotype of Brca1+/- mammary glands. In Aim 2, I will functionally characterize the differentiation potential, hormone responsiveness, and DNA repair to assess whether Brca1+/- mammary cells possess differentiation blocks and deficient DNA repair and whether such alterations are restricted to mammary populations altered in frequency in Aim 1. Finally, in Aim 3, I will take advantage of a novel mouse model developed in the Brugge lab of Brca1-associated breast carcinogenesis to test whether distinct mammary epithelial populations have differing tumorigenic capacity. In sum, the experiments proposed herein will greatly advance our knowledge of how the normal mammary gland is organized and how it is altered in the context of Brca1 heterozygosity. In addition, I will be directly addressing the tumorigenic potential of various mammary populations, thus providing direct evidence of the cell-of-origin in this familial cancer syndrome.